| Cucumber Fusarium wilt is a very serious soil-borne disease in cucumber production,and it is devastating.The Fusarium oxysporum f.sp.cucumerinum can survive in the soil for many years in the state of withering spores,which has always been one of the important factors restricting the high quality and high yield of cucumber.At present,there is a lack of convenient,environment-friendly and effective control methods,and the main strategy to control cucumber Fusarium wilt is prevention.Our research team has found that celery extracts of fresh roots and rhizosphere soil have allelopathic effects on FOC,and it already performed sixth chromatographic to isolating and identificating allelochemicals from fresh celery roots and rhizosphere soil.However,the allelopathic effects of extracts from celery rotten roots and rhizosphere soil on FOC and the changes in metabolic pathways and gene expression of cucumber seedlings after induced were not reported.In this study,celery rotten roots and rhizosphere soil as test materials,acetone,ethanol and distilled water as extractants,combined with each other,6 kinds of extracts can be obtained.After twice column chromatography with 6 extracts,mixed obtained fractions and PDA in the plate,respectively,waiting for the culture medium got solidified,inoculated the FOC bacterial cake(6 mm dia)on the plate.Through colony diameter and allelopathy,24 optimal fractions were finally screened from the 6 extracts,and the physiological and biochemical changes in bacteria were determined.In order to find the allelochemicals in celery materials,24 optimal fractions were detected by GC-MS,and the specific substances contained in the fractions were identified.Then,the 24 best fractions were used as inducers to study the induced resistance of cucumber seedlings.Finally sending the cucumber seedling that treated with highest inducing fraction to transcriptomics sequencing analysis.The results of the study were as follows:1.24 optimal fractions were obtained by second time chromatography from 6 kinds of celery rotten root and rhizosphere soil extracts.Compared with the control,all fractions in the screening process can inhibit the growth of Fusarium oxysporum,have significant allelopathic effects on Fusarium oxysporum,and can significantly inhibit the germination of Fusarium oxysporum spores.Among them,the allelopathic effects of the celery rotten root acetone extract and the celery rotten rhizosphere soil ethanol extract were the most significant,with the allelopathic effects of 29.83%?33.65%?40.51%?41.67% and 37.61%?36.78%?41.80%?44.96%,respectively.The germination inhibition rates of spores of Fusarium oxysporum were 50%,53.70%,61.57%,65.28% and 57.87%,57.41%,65.28%,69.44%,respectively.2.24 optimal fractions obtained from the second time chromatography of the extracts of celery rotted roots and rhizosphere soil acted on Fusarium oxysporum,which significantly affected the biochemical metabolism of the bacteria,including the activity of cell wall degrading enzymes(CWDEs: cellulase,pectinase and ?-glucosidase);membrane permeability(MDA content and soluble protein content);fusaric acid content and enzyme activities of antioxidant system(CAT,POD and SOD).The results showed that the activities of CWDEs in bacterial was decreased significantly,which was negatively correlated with allelopathy and positively correlated with allelopathy.After treatment,the content of Fusarium acid was also decreased significantly,which was significantly different from the control,positively correlated with the activity of CWDEs and negatively correlated with allelopathy.After using the optimum fraction to treated Fusarium oxysporum,the content of MDA was increased obviously and the content of soluble protein was decreased,it indicated that the cell membrane of the bacteria was destroyed,and the content of MDA was positively correlated with allelopathy and negatively correlated with soluble protein.As for the antioxidant system in the bacteria,the activities of protective enzymes CAT,POD and SOD decreased significantly after treatment,which showed a significant negative correlation with allelopathy and a significant positive correlation among the three enzymes.3.Using the GC-MS to detected the 24 best fractions that obtained by second time column chromatography of 6 kinds of extracts from celery rotten roots and rhizosphere soil.Among them,30 substances were identified in the acetone extracts of celery rotten root;22substances were identified in the ethanol extracts of celery rotten root;a total of 9substances were identified in the distilled water extracts of celery rotten root and 12 substances were identified in the acetone extracts of celery rotten rhizosphere soil;the ethanol extracts of celery rotten rhizosphere soil was identified 23 substances;11substances were identified in the distilled water extracts of celery rotten rhizosphere soil.Among them,in addition to the main substance of the distilled water extracts of celery rotten rhizosphere soil was alcohols,the other 5 extracts main substances were esters,organic acids and nitrogen-containing compounds.Combined with the allelopathic effects of each extract on Fusarium oxysporum,esters,organic acids and nitrogen-containing compounds should be the main allelopathic substances,which laid the foundation for the research of allelopathic substances in the future.4.The induced resistance of cucumber seedlings to cucumber Fusarium wilt can be improved after being treated with 24 optimal fractions.Among them,the 4 best fractions of the second time column chromatography of acetone extracts from celery rotten roots and the 4 best fractions of the second time column chromatography of ethanol extracts from celery rotten rhizosphere soil had the most significant control effects on cucumber fusarium wilt,which were 78.95%,78.95%,86.84%,89.47% and 81.58%,81.58%,84.21%,84.21%respectively.The highest control effect was 89.47%,corresponding to the best fraction is RRA102,so RRA102 was selected as the strongest fraction.5.Illumina Nova Seq 6000,was applied for transcriptome analysis of cucumber seedlings induced by the strongest fraction RRA102.Through differential expression analysis,a total of 322 differentially expressed genes(DEGs)were identified,of which 152 were up-regulated and 170 were down-regulated.With GO function annotation classifications,a total of 216 genes were divided into different function categories.The mainly involved biological process,cell structure,enzyme activity and many other physiological and biochemical processes.KEGG enrichment analysis showed that a total of 103 genes were annotated to 63 metabolic pathways with nitrogen metabolism,ascorbic acid and aldonic acid metabolism,amino acid biosynthesis,amino sugar and nucleotide sugar metabolism,and plant hormone signaling pathway significantly enriched,the above pathways are related to plant disease resistance.The results showed that cucumber seedlings stimulated their own defense system after induction treatment,thereby effectively inhibited the occurrence of cucumber fusarium wilt.This study provided a theoretical basis for further green and effective prevention of control the cucumber fusarium wilt,and identifying the resistance genes. |
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